Environmental dissimilarity and geographic distance are recognized as two major factors influencing beta diversity. However, whether and how beta diversity of marine bacteria in a vertical dimension would be influenced by water mixing and stratification remains unclear. Here, we collected 78 samples from surface and chlorophyll maximum (CM) layers along a transect of Kuroshio region east of Taiwan across seasons. We applied the variation portioning to disentangle the relative importance of environmental dissimilarity, vertical distance between sampling depths and the stratification strength in determining the vertical beta diversity of the paired surface and CM bacteria communities in water mixing periods versus stratification periods. Our results suggested that during stratification periods, the vertical beta diversity was significantly explained by environmental dissimilarity, which was determined by strength of stratification in combination of vertical distance. Whereas in mixing periods, environmental dissimilarity alone was the most important factor explaining vertical beta diversity. Vertical beta diversity in the Kuroshio east of Taiwan was structured by different mechanisms, depending on the strength of mixing versus stratification of water column. Climate changes affecting on ocean mixing regime can have profound effects on vertical beta diversity of microbes, with potential impacts on their ecosystem functioning.